The human colon harbors an ecosystem with a rich variety of commensal bacteria that suppress populations of pathogenic bacteria such as Clostridium difficile, the cause of antibiotic-associated colitis. C. difficile disease results when antibiotics damage bacteria that suppress this pathogen, allowing toxigenic strains to establish significant populations in the colon. Initial data from the Pl's lab strongly suggest that a specific phylogenetic group of human commensals is important for the suppression of C. difficile. The goal of the work proposed in this application is to pursue further data to either support or refute the hypothesis that Clostridium coccoides and related organisms protect humans from C. difficile disease. The Specific Aims are 1) to observe the correlation between the population size of C. difficile and the size and complexity of the C. coccoides group in patients treated with antibiotics, 2) to compare the population size of C. difficile in germfree mice with that in gnotobiotic mice colonized with these human commensals and 3) to determine how these commensals suppress C. difficile in a continuous-flow culture model of the colonic ecosystem. Work related to Specific Aim 1 will involve a prospective study of patients on antibiotics. Before and after antibiotic treatment, fecal specimens will be obtained for assessment of C. difficile population size (quantitative cultures) and for determination of the composition of fecal biota. Ribosomal DNA in these specimens will be amplified with PCR, then studied by using 1) high throughput sequencing and phylogenetic analysis of obtained sequences, 2) denaturation gradient gel electrophoresis and 3) a novel 16S rDNA photolithography chip. Because there is not an adequate basis for a power calculation in Specific Aim 1, data obtained might not answer the primary question, but would allow a valid power calculation to be performed to support further study. For Specific Aim 2, germfree mice will be monoassociated with C. difficile followed by either 20 C. coccoides-related organisms, 20 C. leptum-related organisms, or no further biota. C. difficile population size will be followed by quantitative cultures. The mechanisms of suppression (Specific Aim 3) will be determined in a Freter continuous-flow culture model of the large bowel, with emphasis being placed on competition for metabolic substrates.